The long-term goal of this research proposal is to understand how changes in the expression, or regulation of calcium channels in osteoblasts affect bone metabolism. Recent research indicating that the activation of calcium channels in osteoblasts is a common feature of many bone regulatory factors suggests a significant role for intracellular calcium signalling in the control of bone remodeling. However, the molecular structure and cellular regulation of osteoblast calcium channels remain largely undefined. Previous studies have demonstrated that three isoforms of calcium channels are expressed in osteoblast-like osteosarcoma cells (alpha1S, alpha1C and alpha1D), and that parathyroid hormone selectively activates the alpha1D isoform. The objective of this study is to more fully characterize the structure, function and regulation of the calcium channel alpha1D isoform in osteoblasts. The Specific Aims are to: 1) determine the molecular structure of the alpha1D isoform; 2) examine the temporal and hormonal regulation of alpha1D gene expression during osteoblast differentiation; 3) define the mechanism of alpha1D activation by parathyroid hormone; 4) determine the specific functional effects mediated by the alpha1D signaling pathway; and 5) examine the regulation of alpha1D activity by other hormones and drugs. Calcium channel cDNA clones will be isolated, sequenced and used for the quantitative analysis of mRNA levels. Calcium channel activity will be assessed (using a fluorescent probe) to measure the level of intracellular calcium, as well as a radioisotope uptake assay to measure calcium influx. Specific calcium channel antagonists, as well as antisense techniques, will be used to examine the functional roles and hormonal regulation of osteoblast calcium channels. It is suggested that the results of the proposed research study will provide novel and important information about the molecular structure and cellular regulation of osteoblast calcium channels and their role in bone metabolism, as well as providing insight into the cellular mechanisms of action of bone regulatory hormones. These insights are critical to an understanding of the events that regulate bone remodeling and, ultimately, for the development of effective therapeutic approaches for the treatment of bone diseases, including osteoporosis.